Tripeptide

ABSTRACT

The new blocked tripeptide Y-(N w  -R&#39;)Arg-Pro-Gly-R wherein R is .[.hydroxy, methoxy or.]. amino, R&#39; is a suitable blocking group and Y is hydrogen or an easily removable protective group has been found to be a valuable intermediate for the preparation of large peptide chains, such as for instance, the decapeptide Gn-RH.

DETAILED DESCRIPTION OF THE INVENTION

Recent discovery of the aminoacid sequence of the gonadotropin(Gn)-relasing hormone (RH) has made it highly desirable to produce thissubstance on a practical scale in a purity sufficient to use thesubstance therapeutically in instances of hormone deficiences andpossibly as a regulating agent for the ovulation cycle in femalewarm-blooded animals. For instance, it has been found that small dosesof Gn-RH, administered by intravenous injections to female sheep in theanestrus cycle, produces ovulation. The formula of the Gn-RH has beenidentified with the aminoacid sequencepyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH₂ but in order to make such alarge molecule from simple, single aminoacids, a considerable number ofsteps including several condensation reactions are required. In order toassure such condensations to take place at the desired sites, otheractive sites or functional groups on the molecule might be convenientlyprotected by some groups that can be removed at will.

A relatively simple method has now been devised to produce the desiredaminoacid chain in surprisingly good yields. The new methods involves aminimum of group-protecting and -removal reactions for such protectivegroups and employs a number of new intermediates which are importantstepping stones for making Gn-RH and other peptides.

For the purpose of the present disclosure, it is to be understood thatall aminoacids used herein are in their optically active L-form exceptfor glycine.

It has now been found that in order to prepare the decapeptide referredto above, various new intermediates are necessary to accomplish the mostpractical synthesis for such large peptides. These intermediates requireso-called protecting groups on those functional groups that mayinterfere with the desired coupling reaction that extends the peptidechain to a larger number of aminoacids. Such a protective group has tobe found sufficiently strongly to the aminoacid's functional group thatit will remain attached thereto when the blocking group at the N.sup.α-position is removed in order to make that site reactive for couplingwith a chain-extending aminoacid. By properly selecting these protectivegroups, other N.sup.α -blocked aminoacids can be attached to the N.sup.α-position of the present polypeptide and all protective groups can beremoved at the point where the desired chain is completed.

The present invention is directed to a small peptide chain that containsa blocking group that fulfills the above requirement. It is thereforethe main object of the present invention to provide a tripeptide of theformula Y-(N.sup.ω -R')Arg-Pro-Gly-.[.R.]. .Iadd.NH.Iaddend.wherein .[.Rrepresents hydroxy, methoxy or the amino group,.]. R' is a blockinggroup that protects the imino group of the arginine moiety and can beremoved by a simple chemical step that leaves the aminoacid bondsintact, and Y is hydrogen or a protective group that can be removed by asimple, mild chemical treatment which leaves the remainder of themolecule intact. More specifically, where Y is different from hydrogen,it is tert.-butoxycarbonyl (BOC), o-nitrophenylsulfenyl (NPS),2-(diphenyl)isopropyloxycarbonyl, benzyloxycarbonyl (CBZ) or phthalyl.R' may be nitro, p-nitrobenzyloxycarbonyl,tetrachloroisopropyloxyphthaloyl or p-tolylsulfonyl (tos.). Among these,nitro or tos. groups are preferred because they are removable by asimple treatment with catalytic hydrogenation or hydrofluoric acid.Others mentioned must be removed by more complex reactions.

In a simple embodiment, the new compounds of the present invention areprepared by reacting BOC-proline p-nitrophenyl ester with glycinamide.[.or glycine methyl ester.]., preferably by using an excess of thelatter, and the obtained protected dipeptide is converted toPro-Gly-.[.R.]. .Iadd.-NH₂ .Iaddend. by a mild acid treatment. The freedipeptide is then reacted with BOC-(N.sup.ω -R')-Arg in the presence ofdicyclohexylcarbodiimide and an inert solvent. After removing the formeddicyclohexylurea, the mixture is stripped of the solvent and the residueis purified by chromatography. The N.sup.α -BOC group can be removedeasily by a mild acid treatment in an inert organic medium, whileretaining the blocking group R'. .[.Where R is methoxy, the free acid isobtained by hydrolysis in known manner..].

In order to illustrate the method for obtaining the compounds of thepresent invention, reference is made to the following examples whichare, however, not to be interpreted as limiting the scope of thisinvention in any respect.

Example 1

A solution of 514 mg. of prolylglycinamide in 8 ml. of pyridine is mixedat room temperature with 619 mg. of dicyclohexylcarbodiimide and 106.2mg. of N.sup.α -benzyloxycarbonyl-N.sup.ω -nitroarginine. After 16hours, the formed dicyclohexylurea is filtered off and the filtrate isevaporated resulting in an oil. This oil is placed on a chromatographiccolumn containing 35 g. of silica gel using 5% methanolic chloroform asthe solvent. Elution of the column with 5% methanolic chloroform removessome of the impurities contained in the crude product. The pure materialis eluted when the methanol concentration is increased to 15%. Bycombining the appropriate fractions and evaporation of the solvent,1.319 g. (87% of theory) of pure CBZ-(N^(W) -NO₂)Arg-Pro-Gly-NH₂ ofundefined melting point is obtained. The material produces a correctelemental analysis and its NMR spectrum is consistent with the assignedstructure. The compounds show [α]_(D) ²⁵ -25.4° (c.-1, DMF).

Similarly, the tripeptide is made wherein the CBZ-group is replaced withthe BOC-group. However, this material again does not crystallize.

.[.When the above Pro-Gly-NH₂ is replaced by an equimolar amount ofPro-Gly-OCH₃, the same reaction sequence yields the N.sup.α,N.sup.ω-diprotected Arg-Pro-Gly-OCH₃ which is hydrolyzed at room temperature in6 hours with one molar equivalent of 1 N aqueous sodium hydroxide usinga mixture of dimethylformamide/dioxan 1:1 as the solvent for thediprotected Arg-Pro-Gly-OCH₃ to Arg-Pro-Gly-OH carrying the selectedblocking groups in the N.sup.α -positions of Arg..].

Example 2

A solution of 1.013 g. of CBZ-(N.sup.ω -NO₂)Arg-Pro-Gly-NH₂ from Example1 in 8 ml. of acetic acid is treated with 8 ml. of 32% hydrobromic acidin acetic acid. After one hour, the solution is added to ether and theprecipitate is separated, washed five times by suspending it in etherand decanting the supernatant from the solid. The solid is then treatedin methanol with an ion exchange resin in its OH-form and the resultingsuspension is filtered. The resin is washed with 10% acetic acid inmethanol and the combined wash liquor and filtrate is evaporated to asolid of undefined melting point. The elemental analysis confirms theexpected structure (N.sup.ω -NO₂)Arg-Pro-Gly-NH₂ which shows a singlespot on TLC with R_(f) 0.15 in 15% methanol/chloroform.

By replacing (CBZ)-(N.sup.ω -NO₂)Arg-Pro-Gly-NH₂ with the correspondingBOC- protected tripeptide amide or the corresponding N.sup.ω -tos.analogues from Example 1, the above procedure yields (N.sup.ω-NO₂)Arg-Pro-Gly-NH₂ or (N.sup.ω -tos.)Arg-Pro-Gly-NH₂, respectively. Inall instances, the N.sup.α -deprotection step produces a yield of >90%of theory.

The new tripeptide is extremely useful as an intermediate for makinglonger peptide chains, as for instance in Gn-RH and is particularly wellsuited as a precursor in such a synthesis because of its opticalconfiguration with Pro and Arg both being present in the L-form, and theretention of the protective group in the .[.orginine.]. .Iadd.arginine.Iaddend.moiety during the deblocking of the N.sup.α -position andduring any desired subsequent coupling reactions with other aminoacids.During such deblocking and coupling reactions, the new intermediate ischemically and optically stable, i.e., no racemization takes place.

I claim:
 1. The optically active L-form of the tripeptide Y-(N.sup.ω-R')Arg-Pro-Gly-R wherein R is .[.hydroxy, methoxy or.]. amino, R' isnitro, p-nitrobenzyloxycarbonyl, tetrachloroisopropyloxyphthaloyl orp-tolylsulfonyl, and wherein Y is hydrogen, tert.-butoxycarbonyl,o-nitrophenylsulfenyl, 2-(diphenyl)isopropyloxycarbonyl,benzyloxycarbonyl or phthalyl.
 2. The compounds of claim 1 wherein R is.[.hydroxy, methoxy or.]. amino, R' is p-toluenesulfonyl,p-nitrobenzyloxycarbonyl, tetrachloroisopropoxyphthaloyl, or nitro and Yis hydrogen, tert.-butoxycarbonyl, o-nitrophenylsulfenyl, phthalyl,benzoyloxycarbonyl or 2-(diphenyl)isopropyloxycarbonyl.
 3. The compoundof claim 2 wherein R is amino, R' is nitro and Y is hydrogen.
 4. Thecompound of claim 2 wherein R is amino, R' is p-toluenesulfonyl and Y ishydrogen.
 5. The compound of claim 2 wherein R is amino, R' isp-toluenesulfonyl and Y is benzyloxycarbonyl.
 6. The compound of claim 2wherein R is amino, R' is toluenesulfonyl and Y is tert.-butoxycarbonyl.7. The compound of claim 2 wherein R is amino, R' is nitro and Y isbenzyloxycarbonyl. .[.8. The compound of claim 2 wherein R is amino, R'is nitro and Y is trert.-butoxycarbonyl. .].